The identification of patients at high risk of recurrence is one of the most important assessments in the clinical management of breast cancer. Sentinel lymph node status is the current gold standard for clinical discrimination between good and poor prognosis and for deciding if patients receive adjuvant chemotherapy following surgery. However, ~30% of women with node-negative status experience recurrence within 10 years of initial treatment, while ~25% of women with node-positive status do not. Accordingly, there is a need for additional prognostic methods to assess recurrence and survival in breast cancer patients. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have been validated at the highest level of evidence (LOE-1) as clinical biomarkers of prognosis in breast cancer. The American Society of Clinical Oncology recommends using uPA and PAI-1 levels in breast tumors for the routine assessment of prognosis in patients with newly diagnosed breast cancer and for deciding whether node-negative breast cancer patients can forgo adjuvant chemotherapy. The sole validated method for quantifying uPA and PAI-1 levels in breast tumor tissue is a colorimetric ELISA assay requiring a minimum of 300 mg of tumor tissue. Generally, only open (excisional) biopsy will yield 300 mg of tissue, while the more common vacuum-assisted core needle biopsies yield d 110 mg of tumor tissue. The 300 mg requirement also places a restrictive lower limit on the size of tumors that can be evaluated. Accordingly, the availability of a reliable method to accurately quantify uPA and PAI-1 levels in d100 mg of biopsy tissue would have a profound impact on the clinical management of breast cancer. The objective of the studies outlined in this proposal is to develop technologies to rapidly and accurately quantify levels of uPA and PAI- 1 in small tissue specimens (10-100 mg) obtained by fine needle core biopsies of breast tumors. Our central hypothesis is that the combined use of an innovative pressure cycling technology to solubilize tissue proteins and a novel and highly sensitive immunoassay format, called immunoliposome-polymerase chain reaction (ILPCR), developed in our laboratory will achieve the level of quantification for uPA and PAI-1 stated in our objective. The rationale for these studies is that their successful completion will provide clinicians with more reliable means to determine those node-negative patients most likely to benefit from adjuvant chemotherapy following surgery for beast cancer. Accordingly, our project proposes the following three specific aims. Aim 1 is to develop and optimize a method to rapidly and quantitatively extract soluble uPA and PAI-1 from breast tumor tissue using rapid pressure-cycling technology. Aim 2 is to develop and optimize ILPCR assays that can accurately quantify uPA and PAI-1 from 10-100 mg of breast tumor tissue with an assay sensitivity and specificity of e95%. Aim 3 is to validate the rapid pressure-cycling extraction method and ILPCR assays for uPA and PAI-1 against the gold standard ELISA assay by using tissue obtained from the same breast tumor for both assays. These studies are innovative because they will overcome a critical barrier to the use of uPA and PAI-1 for the prognostic evaluation of breast tumors. This research will also improve clinical practice because it will provide clinicians with more reliable means to determine those node-negative patients most likely to benefit from adjuvant chemotherapy, particularly those patients with hormone receptor-negative and node-negative grade 2 tumors who are borderline candidates for chemotherapy. Finally, this research is relevant to Veterans and the VA health care system because it will improve the survival and quality of life of breast cancer patients. It will also facilitate the detection and prognostic assessment of breast tumors at earlier stages where clinical options are greatest and where treatment outcomes are most likely to be favorable.